This study concerns an investigation of the role of Ca++ in the regulation of GABA binding to GABAAlpha and GABABeta recognition sites located in the synaptic membranes of rat brain. At 37 C the binding of [3H]-GABA to GABABeta recognition sites is dramatically stimulated by Ca++ with a Ka of about 10 to the -5M while the binding to GABAAlpha recognition sites is only slightly but significantly enhanced by Ca++ with a Ka of about 5x10 to the -7 M. The Ca++ effect on GABAAlpha recognition sites is temperature dependent and requires calmodulin but involves neither phospholipase A2 nor Ca++ dependent proteases. Only GABAAlpha recognition sites are linked to benzodiazepine recognition sites and also this interaction is modulated by Ca++ at physiological ion concentrations. Diazepam and low MuM Ca++ cause the appearance of a high affinity binding site for GABAAlpha recognition sites. The number of GABABeta recognition sites measured at 37 C is about 70% higher than that measured at 4C. This temperature-dependent increase in the number of GABABeta recognition sites is calmodulin independent, probably Ca++ dependent proteases are operative. We have also studied whether GABAAlpha and benzodiazepines recognition sites located on C6-glioma and neuroblastoma NB2 cells are linked to some other transducer, for instance phospholipase A2. In C6-glioma but not NB2a cells, prelabeled with 14C-arachidonic acid, a GABA agonist muscimol stimulates the release of 14C-arachidonic acid, a GABA agonist muscimol stimulates the release of 14C-arachidonic acid. This increased release is due to an activation of phospholipase A2. This process is blocked by bicuculline, a classical GABAAlpha receptor blocker but not by inhibitors of Cl- channel or GABA uptake blockers. The phospholipase A2 activation by muscimol is potentiated by several benzodiazepines, but not by clonazepam. Analyses of the radioactive metabolites released by HPLC reveal that a substantial amount of prostaglandin D2 is formed when muscimol is supplemented with diazepam. Prostaglandin D2 may be a neuromodulator which serves as a linkage of glial cells to neuronal function. We are currently investigating the possibility of the involvement of glial benzodiazepine receptors in the regulation of GABA function mediated through the formation of certain classes of prostaglandins.